Infant nutrition for improving fatty acid composition of brain membranes

ABSTRACT

The present invention relates to infant nutrition, in particular to infant nutrition comprising special lipid globules for improvement of the fatty acid composition in brain membranes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 13/635,389, filed Nov. 28, 2012, which is the National Phase ofInternational Patent Application No. PCT/NL2011/050187, filed Mar. 17,2011, published on Sep. 22, 2011 as WO 2011/115490 A1, which claimspriority to International Patent Application No. PCT/NL2010/050142,filed Mar. 17, 2010. The contents of these applications are hereinincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to nutrition comprising special lipidglobules, in particular to infant nutrition, for improvement of thefatty acid composition in brain membranes.

BACKGROUND OF THE INVENTION

Breast-feeding is the preferred method of feeding infants. However,there are circumstances that make breast-feeding impossible or lessdesirable. In those cases infant formulae are a good alternative. Thecomposition of modern infant formulae is adapted in such a way that itmeets many of the special nutritional requirements of the fast growingand developing infant.

Still it seems that improvements can be made towards the constitution ofinfant milk formulae. Early nutrition administered during the specificperiod of infancy when rapid growth and development of the body occurshas an imprinting or programming effect and therefore has long termmetabolic consequences. Breast fed infants have a decreased chance ofbecoming obese later in life. Breast-fed infants score better on visualand developmental tests than do formula-fed infants and have an improvedneurodevelopment compared to formula fed infants. Also long term linkshave been reported between breast milk feeding and cognitive ability orneurological status later in life.

This difference in neurodevelopment between breast and bottle fedinfants has mainly been attributed to the presence of long chainpolyunsaturated fatty acids (LC-PUFA) such as docosahexaenoic acid (DHA)and arachidonic acid (ARA) in breast milk. Most current infant milkformulae therefore now also comprise such LC-PUFA. It has also beenfound that such LC-PUFA are better incorporated into membranes when theyare present in the diet in the form of phospholipids instead oftriglycerides.

WO 2008/005033 discloses infant formula comprising fat, protein,carbohydrate, vitamins, and minerals, including gangliosides,phospholipids, (lipid-bound) sialic acid, docosahexaenoic acid, andarachidonic acid for early brain development such as accelarating neuralmigration.

WO 2005/051091 discloses a specific blend of glycerophospholipids incombination with sphingomyelin and/or cholesterol, which blend resemblesthat of human breast milk and is present as a fat globule for use in themanufacture of infant formulae. The blend is claimed to be beneficialfor the development of cognitive and vision functions of the fetus,infants and children.

WO 2009/057121 discloses a method for improving, promoting ormaintaining the development of brain and retina in an infant comprisingadministering a composition comprising at least one triglyceride, atleast one phospholipid and at least one long chain poly-unsaturatedfatty acid (LC-PUFA); wherein at least about 1% of the LC-PUFA in thecomposition is conjugated to said at least one phospholipid.

WO 2009/051502 discloses the use of one or more complex lipids includinggangliosides to achieve particular health benefits including maintainingor increasing cognitive development or maintaining or increasing growthin a foetal, infant or child subject.

US 2008-292724 discloses that upon administration of a composition thatcomprises: a) a lipid fraction comprising at least one ofdocosahexaenoic acid (DHA), docosapentaenoic acid (DPA) andeicosapentaenoic acid (EPA); b) a protein fraction comprisingproteinaceous material from non-human origin which provide at leastcysteine and/or taurine; and c) a mineral fraction comprising at leastone of manganese and molybdene, the health of these persons improves.Membrane function of cells improves, which allows efficient treatment ofdisorders, amongst which cognitive dysfunction and other diseases of thenervous system, neuropathies

WO 2009/138680 discloses that the presence of at least 30% milk fat inconjunction with a vegetable oil in infant nutrition can be used amongstothers to increase DHA accumulation in brain membranes, and ameloriatingbrain development and cognitive function. Optionally milk phospholipidsare present.

WO 2008/081934 discloses an agent for facilitating the development ofthe brain in an infant, which comprises an effective amount of amilk-derived phospholipid or a sphingomyelin.

WO 2007/073193 discloses that in IMF with low levels of n6 PUFA,necessary to prevent obesity later in life, the incorporation of thesmall amount of n6 (LC-)PUFA into neurological cell membranes is moreefficient by providing lipidic membrane components such as cholesterol,phospholipids and/or sphingolipids.

Benoit et al, 2010, Food Chem, 120:684-691, disclose that PC is anefficient carrier for DHA accretion in membranes and that in thisrespect also the specific structurisation of most PL in human milk, inthe native milk fat globule membrane, which cannot be copied in infantformula, may be of functional significance for the infant.

Vickers et al, 2009, Nutr. Res., 29:426-435, disclose that high levelsof complex lipids derived from milk improved parameters related tocognition. The amount of phospholipids based on total fat intakeexceeded 6 wt. %, the phospholipids were administered separately viagavage, and also docosahexaenoic was supplemented to the complex lipidsupplement.

SUMMARY OF THE INVENTION

Using rodent animal models the inventors found that even after a longperiod during which all the animals were on the same Western style diet,the effects of a previous early diet administered during infancy werestill present with regard to the fatty acid profile of the brainmembranes. Since fatty acid accretion of the brain and turnover in thebrain is a continuous process throughout life, it was unexpected thatsuch long term early diets effects were observed. The most surprisingfinding however, was that this effect was observed with infancy dietswith a similar fat composition, only differing in the architecture ofthe dietary lipid globules. Effects on long term brain membrane fattyacid composition were observed regarding the presence and location ofphospholipids and the size of the lipid globules. Phospholipids weremost effective when located in the coating, i.e. outer layer, of thelipid globule, instead of being present as a free ingredient. Bestresults were obtained with an early diet comprising large lipid globulescoated with phospholipids resulting in a long term increased percentageof PUFA and LC-PUFA in brain membranes, in particular DHA, indicativefor increased membrane fluidity. Preferably, the lipid globules have tobe both surrounded by a coating comprising phospholipids and increasedin size in order to see improved long term effect on brain fatty acidcomposition compared to lipid globules as present in standard IMF.

This effect was consistently found in several independent experiments.In other animal experiments a direct effect of size and coating of lipidglobules on brain membrane fatty acids was observed, even after 5 daysof feeding in a young animal. Increased membrane fluidity, LC-PUFAcontent and n3/n6 PUFA ratio in brain membranes are known to becorrelated to improve cognitive and behavioral performance. Hence thepresent invention can be used to improve these performances or to treatand/or prevent cognitive or behavioral disorders.

Cognitive improvement was indeed demonstrated in animals havingadministered the composition of the present invention, as demonstratedby a short version of the Morris water maze test.

The present invention therefore relates to nutrition, in particularinfant nutrition, comprising lipid in the form of lipid globules, coatedwith polar lipids including phospholipids, and that are preferably largein size, for use in the development of cognitive or behaviouralperformances, including fine motor skills and visual acuity.

DETAILED DESCRIPTION

The present invention thus concerns a method for i) increasing brainmembrane fluidity, ii) increasing brain membrane PUFA, iii) increasingbrain membrane LC-PUFA, iv) decreasing ratio of brain membrane n6/n3LC-PUFA, v) decreasing ratio of brain membrane n6/n3 PUFA, vi)increasing brain membrane n3 PUFA, vii) increasing brain membrane n3LC-PUFA and/or viii) increasing brain membrane DHA, in a human subject,by administration of a nutritional composition comprising

a) 10 to 50 wt. % vegetable lipids based on dry weight of thecomposition, and

b1) 0.5 to 20 wt. % phospholipids based on total lipid and/or

b2) 0.6 to 25 wt. % of polar lipids based on total lipids, wherein polarlipids are the sum of phospholipids, glycosphingolipids and cholesterol,

and said composition comprising lipid globules with a core comprisingsaid vegetable lipids and a coating comprising said phospholipids orpolar lipids.

In one aspect present invention concerns a method for altering brainmembrane fatty acid composition.

In one embodiment the present invention concerns a method for alteringbrain membrane fatty acid composition selected from the group consistingof i) increasing brain membrane fluidity, ii) increasing brain membranePUFA, iii) increasing brain membrane LC-PUFA, iv) decreasing ratio ofbrain membrane n6/n3 LC-PUFA, v) decreasing ratio of brain membranen6/n3 PUFA, vi) increasing brain membrane n3 PUFA, vii) increasing brainmembrane n3 LC-PUFA and viii) increasing brain membrane DHA, in a humansubject, by administration of a nutritional composition comprising

a) 10 to 50 wt. % vegetable lipids based on dry weight of thecomposition, and

b1) 0.5 to 20 wt. % phospholipids based on total lipid and/or

b2) 0.6 to 25 wt. % of polar lipids based on total lipids, wherein polarlipids are the sum of phospholipids, glycosphingolipids and cholesterol,

and said composition comprising lipid globules with a core comprisingsaid vegetable lipids and a coating comprising said phospholipids orpolar lipids.

In one embodiment the present method is non-therapeutic.

The present invention can also be worded as the use of a compositioncomprising lipid, or the use of lipid, for the manufacture of anutritional composition for altering brain membrane fatty acidcomposition selected from the group consisting of i) increasing brainmembrane fluidity, ii) increasing brain membrane PUFA, iii) increasingbrain membrane LC-PUFA, iv) decreasing ratio of brain membrane n6/n3LC-PUFA, v) decreasing ratio of brain membrane n6/n3 PUFA, vi)increasing brain membrane n3 PUFA, vii) increasing brain membrane n3LC-PUFA and viii) increasing brain membrane DHA, in a human subject,said nutritional composition comprising

a) 10 to 50 wt. % vegetable lipids based on dry weight of thecomposition, and

b1) 0.5 to 20 wt. % phospholipids based on total lipid and/or

b2) 0.6 to 25 wt. % of polar lipids based on total lipids, wherein polarlipids are the sum of phospholipids, glycosphingolipids and cholesterol,

and said composition comprising lipid globules with a core comprisingsaid vegetable lipids and a coating comprising said phospholipids orpolar lipids.

The present invention can also be worded as a nutritional compositioncomprising

a) 10 to 50 wt. % vegetable lipids based on dry weight of thecomposition, and

b1) 0.5 to 20 wt. % phospholipids based on total lipid and/or

b2) 0.6 to 25 wt. % of polar lipids based on total lipids, wherein polarlipids are the sum of phospholipids, glycosphingolipids and cholesterol,

and said composition comprising lipid globules with a core comprisingsaid vegetable lipids and a coating comprising said phospholipids orpolar lipids

for use in altering brain membrane fatty acid composition selected fromthe group consisting of i) increasing brain membrane fluidity, ii)increasing brain membrane PUFA, iii) increasing brain membrane LC-PUFA,iv) decreasing ratio of brain membrane n6/n3 LC-PUFA, v) decreasingratio of brain membrane n6/n3 PUFA, vi) increasing brain membrane n3PUFA, vii) increasing brain membrane n3 LC-PUFA and viii) increasingbrain membrane DHA, in a human subject.

In one embodiment the present invention is for the prevention and/ortreatment of a disorder associated with decreased brain membranefluidity and/or associated with decreased brain membrane PUFA contentand/or LC-PUFA content. In one embodiment, the disorder is apsychiatric, psychological and/or neurobiological disorder. In oneembodiment the present invention is for amelioration of i) cognitiveperformance, preferably memory performance and/or language developmentperformance, more preferably learning memory performance, ii)behavioural performance, iii) visual acuity, iv) fine motor skills.

In one apect the invention concerns a method for the prevention and/ortreatment of a disorder associated with decreased brain membranefluidity and/or associated with decreased brain membrane PUFA contentand/or LC-PUFA content and/or increased ratio of n6/n3 LC-PUFA, and/orincreased ratio of n6/n3 PUFA by administration to a human subject of anutritional comprising

a) 10 to 50 wt. % vegetable lipids based on dry weight of thecomposition, and

b1) 0.5 to 20 wt. % phospholipids based on total lipid and/or

b2) 0.6 to 25 wt. % of polar lipids based on total lipids, wherein polarlipids are the sum of phospholipids, glycosphingolipids and cholesterol,

and said composition comprising lipid globules with a core comprisingsaid vegetable lipids and a coating comprising said phospholipids orpolar lipids.

In one aspect, the present invention thus concerns a method fortreatment and/or prevention of a disorder selected from the groupconsisting of attention deficiency, ADHD, dyslexia, autism, depression,bipolar depression, anxiety, schizophrenia, obsessive-compulsivedisorder (OCD), bulimia, abuse of alcohol or drugs, borderlinepersonality disorder, panic disorder, social phobia, learningdifficulties, mild cognitive impairment, learning memory impairment,language development impairment, dementia, Alzheimer's disease andParkinson's disease, said method comprising administering to a humansubject a nutritional composition comprising

a) 10 to 50 wt. % vegetable lipids based on dry weight of thecomposition, and

b1) 0.5 to 20 wt. % phospholipids based on total lipid and/or

b2) 0.6 to 25 wt. % of polar lipids based on total lipids, wherein polarlipids are the sum of phospholipids, glycosphingolipids and cholesterol,

and said composition comprising lipid globules with a core comprisingsaid vegetable lipids and a coating comprising said phospholipids orpolar lipids

The present invention can also be worded as the use of a compositioncomprising lipid, or the use of lipid, for the manufacture of anutritional composition for treatment and/or prevention of a disorderselected from the group consisting of attention deficiency, ADHD,dyslexia, autism, depression, bipolar depression, anxiety,schizophrenia, OCD, bulimia, abuse of alcohol or drugs, borderlinepersonality disorder, panic disorder, social phobia, learningdifficulties, mild cognitive impairment learning memory impairment,language development impairment, dementia, Alzheimer's disease andParkinson's disease in a human subject, said nutritional compositioncomprising

a) 10 to 50 wt. % vegetable lipids based on dry weight of thecomposition, and

b1) 0.5 to 20 wt. % phospholipids based on total lipid and/or

b2) 0.6 to 25 wt. % of polar lipids based on total lipids, wherein polarlipids are the sum of phospholipids, glycosphingolipids and cholesterol,

and said composition comprising lipid globules with a core comprisingsaid vegetable lipids and a coating comprising said phospholipids orpolar lipids.

The present invention can also be worded as a nutritional compositioncomprising

a) 10 to 50 wt. % vegetable lipids based on dry weight of thecomposition, and

b1) 0.5 to 20 wt. % phospholipids based on total lipid and/or

b2) 0.6 to 25 wt. % of polar lipids based on total lipids, wherein polarlipids are the sum of phospholipids, glycosphingolipids and cholesterol,

and said composition comprising lipid globules with a core comprisingsaid vegetable lipids and a coating comprising said phospholipids orpolar lipids

for use in treatment and/or prevention of a disorder selected from thegroup consisting of attention deficiency, ADHD, dyslexia, autism,depression, bipolar depression, anxiety, schizophrenia, OCD, bulimia,abuse of alcohol or drugs, borderline personality disorder, panicdisorder, social phobia, learning difficulties, mild cognitiveimpairment learning memory impairment, language development impairment,dementia, Alzheimer's disease and Parkinson's disease in a humansubject.

In one aspect, the present invention concerns a method for ameliorationof i) cognitive performance, preferably memory performance and/orlanguage development performance, more preferably learning memoryperformance, ii) behavioural performance, iii) visual acuity, iv) finemotor skills, in a human subject, said method comprising administeringto a human subject, or in other words by administration to a humansubject of, a nutritional composition comprising

a) 10 to 50 wt. % vegetable lipids based on dry weight of thecomposition, and

b1) 0.5 to 20 wt. % phospholipids based on total lipid and/or

b2) 0.6 to 25 wt. % of polar lipids based on total lipids, wherein polarlipids are the sum of phospholipids, glycosphingolipids and cholesterol,

and said composition comprising lipid globules with a core comprisingsaid vegetable lipids and a coating comprising said phospholipids orpolar lipids.

In one embodiment of this aspect, the method is non-therapeutic.

The invention can also be worded as the use of a composition comprisinglipid, or the use of lipid, for the manufacture of a nutritionalcomposition for amelioration of i) cognitive performance, preferablymemory performance and/or language development performance, morepreferably learning memory performance, ii) behavioural performance,iii) visual acuity, iv) fine motor skills in a human subject, saidnutritional composition comprising

a) 10 to 50 wt. % vegetable lipids based on dry weight of thecomposition, and

b1) 0.5 to 20 wt. % phospholipids based on total lipid and/or

b2) 0.6 to 25 wt. % of polar lipids based on total lipids, wherein polarlipids are the sum of phospholipids, glycosphingolipids and cholesterol,

and said composition comprising lipid globules with a core comprisingsaid vegetable lipids and a coating comprising said phospholipids orpolar lipids.

The present invention can also be worded as a nutritional compositioncomprising

a) 10 to 50 wt. % vegetable lipids based on dry weight of thecomposition, and

b1) 0.5 to 20 wt. % phospholipids based on total lipid and/or

b2) 0.6 to 25 wt. % of polar lipids based on total lipids, wherein polarlipids are the sum of phospholipids, glycosphingolipids and cholesterol,

and said composition comprising lipid globules with a core comprisingsaid vegetable lipids and a coating comprising said phospholipids orpolar lipids

for use in amelioration of i) cognitive performance, preferably memoryperformance and/or language development performance, more preferablylearning memory performance, ii) behavioural performance, iii) visualacuity, iv) fine motor skills in a human subject.

For sake of clarity it is noted that the present invention is defined interms of specific ingredients, hence the vegetable lipids andphospholipids and polar lipids and by the way these ingredients areassembled, hence as phospholipid or polar lipid coated lipid globules ofa certain size. Hence the ingredients and the way they are assembledoverlap.

Throughout the description wherever the phrase ‘the present composition’is used it is to be understood that this refers to the composition thatis used in the method according to the present invention or in otherwords for the use to achieve the specified effect(s).

Lipid Component

The present composition comprises lipid. The lipid provides preferably30 to 60% of the total calories of the composition. More preferably thepresent composition comprises lipid providing 35 to 55% of the totalcalories, even more preferably the present composition comprises lipidproviding 40 to 50% of the total calories. When in liquid form, e.g. asa ready-to-feed liquid, the composition preferably comprises 2.1 to 6.5g lipid per 100 ml, more preferably 3.0 to 4.0 g per 100 ml. Based ondry weight the present composition preferably comprises 10 to 50 wt. %,more preferably 12.5 to 40 wt. % lipid, even more preferably 19 to 30wt. % lipid.

Lipids include polar lipids (such as phospholipids, glycolipids,sphingomyelin, and cholesterol), monoglycerides, diglycerides,triglycerides and free fatty acids. Preferably the composition comprisesat least 75 wt. %, more preferably at least 85 wt. % triglycerides basedon total lipids.

The lipid of the present invention comprises vegetable lipids. Thepresence of vegetable lipids advantageously enables an optimal fattyacid profile, high in (poly)unsaturated fatty acids and/or morereminiscent to human milk fat. Using lipids from cow's milk alone, orother domestic mammals, does not provide an optimal fatty acid profile.Preferably the present composition comprises at least one, preferably atleast two lipid sources selected from the group consisting of linseedoil (flaxseed oil), rape seed oil (such as colza oil, low erucic acidrape seed oil and canola oil), salvia oil, perilla oil, purslane oil,lingonberry oil, sea buckthorn oil, hemp oil, sunflower oil, high oleicsunflower oil, safflower oil, high oleic safflower oil, olive oil, blackcurrant seed oil, echium oil, coconut oil, palm oil and palm kernel oil.Preferably the present composition comprises at least one, preferably atleast two lipid sources selected from the group consisting of linseedoil, canola oil, coconut oil, sunflower oil and high oleic sunfloweroil. Commercially available vegetable lipids are typically offered inthe form a continuous oil phase. When in liquid form, e.g. as aready-to-feed liquid, the composition preferably comprises 2.1 to 6.5 gvegetable lipid per 100 ml, more preferably 3.0 to 4.0 g per 100 ml.Based on dry weight the present composition preferably comprises 10 to50 wt. %, more preferably 12.5 to 40 wt. % vegetable lipid, even morepreferably 19 to 30 wt. %. Preferably the composition comprises 50 to100 wt. % vegetable lipids based on total lipids, more preferably 70 to100 wt. %, even more preferably 75 to 97 wt. %. It is noted thereforethat the present composition also may comprise non-vegetable lipids.Suitable and preferred non-vegetable lipids are further specified below.

Polar Lipids

The present invention comprises polar lipids. Polar lipids areamphipathic of nature and include glycerophospholipids,glycosphingolipids, sphingomyelin and/or cholesterol. More preferablythe composition comprises phospholipids (the sum of glycerophospholipidsand sphingomyelin). Polar lipids in the present invention relate to thesum of glycerophospholipids, glycosphingolipids, sphingomyelin andcholesterol. According to the present invention polar lipids are presentas a coating of the lipid globule. By ‘coating’ is meant that the outersurface layer of the lipid globule comprises polar lipids, whereas thesepolar lipids are virtually absent in the core of the lipid globule. Thepresence of polar lipids as a coating or outer layer of the lipidglobule in the diet administered early in life was found toadvantageously result in increased incorporation of (LC-)PUFA in braincell membranes later in life.

The present composition preferably comprises glycerophospholipids.Glycerophospholipids are a class of lipids formed from fatty acidsesterified at the hydroxyl groups on carbon-1 and carbon-2 of thebackbone glycerol moiety and a negatively-charged phosphate groupattached to carbon-3 of the glycerol via an ester bond, and optionally acholine group (in case of phosphatidylcholine, PC), a serine group (incase of phosphatidylserine, PS), an ethanolamine group (in case ofphosphatidylethanolamine, PE), an inositol group (in case ofphosphatidylinositol, PI) or a glycerol group (in case ofphosphatidylglycerol, PG) attached to the phosphate group.Lysophospholipids are a class of phospholipids with one fatty acylchain. Preferably the present composition contains PC, PS, PI and/or PE,more preferably at least PC.

The present composition preferably comprises phosphospingolipids,preferably sphingomyelin. Sphingomyelins have a phosphorylcholine orphosphorylethanolamine molecule esterified to the 1-hydroxy group of aceramide. They are classified as phospholipid as well as sphingolipid,but are not classified as a glycerophospholipid nor as aglycosphingolipid.

The present composition preferably comprises glycosphingolipids. Theterm glycosphingolipids as in the present invention particularly refersto glycolipids with an amino alcohol sphingosine. The sphingosinebackbone is O-linked to a charged headgroup such as ethanolamine, serineor choline backbone. The backbone is also amide linked to a fatty acylgroup. Glycosphingolipids are ceramides with one or more sugar residuesjoined in a β-glycosidic linkage at the 1-hydroxyl position. Preferablythe present composition contains gangliosides, more preferably at leastone ganglioside selected from the group consisting of GM3 and GD3.

Sphingolipids are in the present invention defined as the sum ofsphingomyelin and glycosphingolipids. Phospholipids are in the presentinvention defined as the sum of sphingomyelin and glycerophospholipids.Preferably the phospholipids are derived from milk lipids. Preferablythe weight ratio of phospholipids:glycosphingolipids is from 2:1 to10:1, more preferably 2:1 to 5:1.

Preferably the present composition comprises phospholipids. Preferablythe present composition comprises 0.5 to 20 wt. % phospholipids based ontotal lipid, more preferably 0.5 to 10 wt. %, more preferably 1 to 10wt. %, even more preferably 1 to 5 wt. %, even more preferably 2 to 10wt. % even more preferably 2 to 5 wt. %, even more preferably 0.5 to 5wt. % and even more preferably 1 to 3 wt. % phospholipids based on totallipid. Preferably the present composition comprises 0.1 to 10 wt. %glycosphingolipids based on total lipid, more preferably 0.5 to 5 wt. %,even more preferably 2 to 4 wt %. Preferably the present compositioncomprises 0.5 to 10 wt. % (glycosphingolipids plus phospholipids) basedon total lipid, more preferably 1.0 to 10 wt. % (glycosphingolipids plusphospholipids), more preferably 0.5 to 6 wt. % (glycosphingolipids plusphospholipids), more preferably 0.5 to 3.5 wt. % (glycosphingolipidsplus phospholipids), more preferably 1.0 to 6 wt. % (glycosphingolipidsplus phospholipids), more preferably 1.0 to 3.5 wt. %(glycosphingolipids plus phospholipids) based on total lipid.

The present composition preferably comprises cholesterol. The presentcomposition preferably comprises at least 0.005 wt. % cholesterol basedon total lipid, more preferably at least 0.02 wt. %, more preferably atleast 0.05 wt. %, even more preferably at least 0.1 wt. %. Preferablythe amount of cholesterol does not exceed 10 wt. % based on total lipid,more preferably does not exceed 5 wt. %, even more preferably does notexceed 1 wt. % of total lipid.

Preferably the present composition comprises 0.6 to 25 wt. % polarlipids based on total lipid, wherein the polar lipids are the sum ofphospholipids, glycosphingolipids, and cholesterol, more preferably 0.6to 12 wt. %, more preferably 0.6 to 6 wt. %, more preferably 1 to 10 wt.%, even more preferably 2 to 10 wt %, even more preferably 3.0 to 10 wt.% polar lipids based on total lipid, wherein the polar lipids are thesum of phospholipids, glycosphingolipids, and cholesterol.

Preferred sources for providing the phospholipids, glycosphingolipidsand/or cholesterol are egg lipids, milk fat, buttermilk fat and butterserum fat (such as beta serum fat). A preferred source forphospholipids, particularly PC, is soy lecithin and/or sunflowerlecithin. The present composition preferably comprises phospholipidsderived from milk. Preferably the present composition comprisesphospholipids and glycosphingolipids derived from milk. Preferably alsocholesterol is obtained from milk. Preferably the polar lipids arederived from milk. Polar lipids derived from milk include the polarlipids isolated from milk lipid, cream lipid, butter serum lipid (betaserum lipid), whey lipid, cheese lipid and/or buttermilk lipid. Thebuttermilk lipid is typically obtained during the manufacture ofbuttermilk. The butter serum lipid or beta serum lipid is typicallyobtained during the manufacture of anhydrous milk fat from butter.Preferably the phospholipids, glycosphingolipids and/or cholesterol areobtained from milk cream. The composition preferably comprisesphospholipids, glycosphingolipids and/or cholesterol from milk of cows,mares, sheep, goats, buffalos, horses and camels. It is most preferredto use a lipid extract isolated from cow's milk. The use of polar lipidsfrom milk fat advantageously comprises the polar lipids from milk fatglobule membranes, which are more reminiscent to the situation in humanmilk. Polar lipids derived from fat milk advantageously improve brainfatty acid composition to a larger extent than polar lipids from othersources. The polar lipids are located on the surface of the lipidglobule, i.e. as a coating or outer layer. A suitable way to determinewhether the polar lipids are located on the surface of the lipidglobules is laser scanning microscopy as given in example 1. Theconcomitant use of polar lipids derived from domestic animals milk andtrigycerides derived from vegetable lipids therefore enables tomanufacture coated lipid globules with a coating more similar to humanmilk, while at the same time providing an optimal fatty acid profile.Suitable commercially available sources for milk polar lipids are BAEF,SM2, SM3 and SM4 powder of Corman, Salibra of Glanbia, and LacProdanMFGM-10 or PL20 from Arla. Preferably the source of milk polar lipidscomprises at least 4 wt. % phospholipids based on total lipid, morepreferably 7 to 75 wt. %, most preferably 20 to 70 wt. % phospholipidsbased on total lipid. Preferably the weight ratio phospholipids toprotein is above 0.10, more preferably above 0.20, even more preferablyabove 0.3. Preferably at least 25 wt. %, more preferably at least 40 wt.%, most preferably at least 75 wt. % of the polar lipids is derived frommilk polar lipids.

Fatty Acid Composition

Herein LA refers to linoleic acid and/or acyl chain (18:2 n6); ALArefers to α-linolenic acid and/or acyl chain (18:3 n3); LC-PUFA refersto long chain polyunsaturated fatty acids and/or acyl chains comprisingat least 20 carbon atoms in the fatty acyl chain and with 2 or moreunsaturated bonds; DHA refers to docosahexaenoic acid and/or acyl chain(22:6, n3); EPA refers to eicosapentaenoic acid and/or acyl chain (20:5n3); ARA refers to arachidonic acid and/or acyl chain (20:4 n6); DPArefers to docosapentaenoic acid and/or acyl chain (22:5 n3). Mediumchain fatty acids (MCFA) refer to fatty acids and/or acyl chains with achain length of 6, 8 or 10 carbon atoms.

LA preferably is present in a sufficient amount in order to promote ahealthy growth and development, yet in an amount as low as possible inview of an unwanted high n6/n3 ratio. The composition thereforepreferably comprises less than 15 wt. % LA based on total fatty acids,preferably between 5 and 14.5 wt. %, more preferably between 6 and 10wt. %. Preferably the composition comprises over 5 wt. % LA based onfatty acids. Preferably ALA is present in a sufficient amount to promotea healthy growth and development of the infant. The present compositiontherefore preferably comprises at least 1.0 wt. % ALA based on totalfatty acids. Preferably the composition comprises at least 1.5 wt. % ALAbased on total fatty acids, more preferably at least 2.0 wt. %.Preferably the composition comprises less than 10 wt. % ALA, morepreferably less than 5.0 wt. % based on total fatty acids. The weightratio LA/ALA should be well balanced ensuring a normal growth anddevelopment. Therefore, the present composition preferably comprises aweight ratio of LA/ALA between 2 and 15, more preferably between 2 and7, more preferably between 4 and 7, more preferably between 3 and 6,even more preferably between 4 and 5.5, even more preferably between 4and 5.

The present composition preferably comprises at least 3 wt. % MCFA basedon total fatty acids, more preferably at least 10 wt. %, even morepreferably 15 wt. %. The present composition advantageously comprisesless than 50 wt. % MCFA based on total fatty acids, more preferably lessthan 40 wt. %, even more preferably less than 25 wt. %.

Preferably the present composition comprises n3 LC-PUFA, since efficientincorporation of n3 LC-PUFA in brain membranes improve fluidity thereof.More preferably, the present composition comprises EPA, DPA and/or DHA,even more preferably DHA. Since a low concentration of DHA, DPA and/orEPA is already effective and normal growth and development areimportant, the content of n3 LC-PUFA in the present composition,preferably does not exceed 15 wt. % of the total fatty acid content,preferably does not exceed 10 wt. %, even more preferably does notexceed 5 wt. %. Preferably the present composition comprises at least0.2 wt. %, preferably at least 0.5 wt. %, more preferably at least 0.75wt. %, n3 LC-PUFA of the total fatty acid content. In one embodiment thepresent composition preferably comprises DHA in an amount of 0.1 to 0.6wt. % based on total fatty acid content.

As the group of n6 fatty acids, especially arachidonic acid (ARA) and LAas its precursor, counteracts the group of n3 fatty acids, especiallyDHA and EPA and ALA as their precursor, the present compositioncomprises relatively low amounts of ARA. The n6 LC-PUFA contentpreferably does not exceed 5 wt. %, more preferably does not exceed 2.0wt. %, more preferably does not exceed 0.75 wt. %, even more preferablydoes not exceed 0.5 wt. %, based on total fatty acids. Nevertheless,since according to the present invention incorporation into brainmembranes is improved, still an advantageous effect on brain membranefluidity can be obtained. Since ARA is important in infants for optimalfunctional membranes, especially membranes of brain tissues, the amountof n6 LC-PUFA is preferably at least 0.02 wt. % more preferably at least0.05 wt. %, more preferably at least 0.1 wt. % based on total fattyacids, more preferably at least 0.2 wt. %. The presence of ARA isadvantageous in a composition low in LA since it remedies LA deficiency.The presence of, preferably low amounts, of ARA is beneficial innutrition to be administered to infants below the age of 6 months, sincefor these infants the infant formulae is generally the only source ofnutrition. In one embodiment the present composition preferablycomprises ARA in an amount of 0.1 to 0.6 wt. % based on total fatty acidcontent.

Preferably in addition to the vegetable lipid, a lipid selected fromfish oil (preferably tuna fish oil) and single cell oil (such as algal,microbial oil and fungal oil) is present. These sources of oil aresuitable as LC-PUFA sources. Preferably as a source of n3 LC-PUFA singlecell oil, including algal oil and microbial oil, is used, since theseoil sources have an advantageous EPA/DHA ratio. More preferably fish oil(even more preferably tuna fish oil) is used as a source of n3 LC-PUFAsince fish oil has a higher EPA concentration. Thus in one embodimentthe present composition further comprises at least one lipid selectedfrom the group consisting of fish oil, marine oil, algal oil, fungal oiland microbial oil.

Process for Obtaining Polar Lipid Coated Lipid Globules

The present composition comprises lipid globules. The lipid globule sizecan be manipulated by adjusting process steps by which the presentcomposition is manufactured. A suitable and preferred way to obtainlipid globules coated with polar lipids is to increase the amount ofpolar lipids compared to amounts typically present in infant formula andto have these polar lipids present during the homogenization process,wherein the mixture of aqueous phase and oil phase is homogenized.Typical amounts of phospholipids/polar lipids in infant formula areabout 0.15 wt. %/0.2 wt. % based on total fat. The amount ofphospholipids is increased to at least 0.5 wt %, more preferably atleast 1.0 wt. % based on total fat or the amount of polar lipids isincreased to at least 0.6 wt. %, more preferably at least 1.0 wt. %based on total fat. In standard infant milk formula the lipid fraction(usually comprising vegetable fat, a small amount of polar lipids andfat soluble vitamins) is mixed into the aqueous fraction (usuallycomprising water, skimmed milk, whey, digestible carbohydrates such aslactose, water soluble vitamins and minerals and optionallynon-digestible carbohydrates) by homogenization. If no homogenizationwas to take place, the lipid part would cream very quickly, i.e.separate from the aqueous part and collect at the top. Homogenization isthe process of breaking up the fat phase into smaller sizes so that itno longer quickly separates from the aqueous phase but is maintained ina stable emulsion. This is accomplished by forcing the milk at highpressure through small orifices.

The process comprises the following steps:

1 Mixing Ingredients

The ingredients of the composition are mixed, e.g. preferably blended.Basically a lipid phase, comprising the vegetable lipids, and an aqueousphase are added together. The ingredients further comprise polar lipids,more preferably phospholipids. The ingredients of the aqueous phase maycomprise water, skimmed milk (powder), whey (powder), low fat milk,lactose, water soluble vitamins and minerals. Preferably the aqueousphase comprises non-digestible oligosaccharides. Preferably the aqueousphase is set at a pH between 6.0 and 8.0, more preferably pH 6.5 to 7.5.

Preferably the polar lipids, in particular the phospholipids, arederived from milk. Advantageously, having polar lipids present in theaqueous mixture before homogenization results in an efficient coating ofthe lipid globules, consisting essentially of triglycerides, with acoating of polar lipids. Preferably the lipid phase comprises 50 to 100wt. % vegetable lipids based on total weight of the lipid phase. Insteadof in the aqueous phase, the polar lipids, more preferably thephospholipids, may also be present in the lipid phase or in both phases.Alternatively the polar lipids may be added separately to an aqueous andlipid phase. Preferably, the weight ratio of phospholipid to total lipidis from 0.5 to 20 wt. %, more preferably from 0.5 to 10 wt. %, even morepreferably 3 to 8 wt. %. Preferably the weight ratio of polar lipids tottotal lipid is 0.6 to 25 wt. %, more preferably from 0.6 to 12 wt. %

The aqueous and lipid phase are preferably heated before addingtogether, preferably at a temperature of 40° C. to 80° C., morepreferably 55° C. to 70° C., even more preferably 55° C. to 60° C. Themixture is also kept at this temperature and blended. A suitable way forblending is using an Ultra-Turrax T50 for about 30-60 s at 5000-10000rpm. Subsequently demi-water may be added to this blend, to obtain thedesired dry matter %. A desired dry matter % is for example 15%.Alternatively, the lipid phase is injected to the aqueous phaseimmediately prior to homogenization. Minerals, vitamins, and stabilizinggums may be added at various points in the process depending on theirsensitivity to heat. Mixing can for instance be performed with a highshear agitator. In the process of the present invention, skimmed milk(casein) is preferably not present in this step and added to thecomposition after homogenization of the fat fraction into the aqueousfraction (comprising compounds such as whey, whey protein, lactose).

2 Pasteurization

Preferably the mixture is then pasteurized. Pasteurization involves aquick heating step under controlled conditions which microorganismscannot survive. A temperature of 60 to 80° C., more preferably 65 to 75°C., held for at least 15 s, usually adequately reduces vegetative cellsof microorganisms. Several pasteurization methods are known andcommercially feasible. Alternatively this step can also be performedbefore mixing as in step 1 and/or be replaced by the heating step to 60°C. in step 1.

3 Homogenization

Subsequently the optionally pasteurized mixture comprising vegetablelipids, polar lipids and an aqueous phase is homogenized. Homogenizationis a process which increases emulsion uniformity and stability byreducing the size of the lipid globules in the formula. This processstep can be performed with a variety of mixing equipment, which applieshigh shear to the product. This type of mixing breaks the lipid globulesinto smaller globules. The mixture obtained is preferably homogenized intwo steps, for example at 250 to 50 bar, respectively, so a totalpressure of 300 bar in order to obtain small, stable lipid globules.

In case the size of the lipid globules is preferred to be larger thehomogenization steps are performed under much lower pressures. Forexample 60° C. at 5 to 100, preferably 30-100, bar and 5 to 50 barrespectively, with a total pressure of 35 to 150 bar. Alternatively, themixture obtained is preferably homogenized in two steps at a lowertemperature, between 15 and 40° C., preferably about 20° C. at 5 to 50and 5 to 50 bar respectively, with a total pressure of 5 to 100 bar.This is remarkably lower than standard pressures, which typically are250 to 50 bar, respectively, so a total pressure of 300 bar. It will bedependent on the specific homogenizer used, which pressure to apply. Asuitable way is to use a pressure of 100 bar in the first step and 50bar in the second step in a Niro Suavi NS 2006 H Homogenizer at atemperature of 60° C. A suitable way is to use a pressure of 5 bar inthe first step and 20 bar in the second step in a Niro Suavi NS 2006 HHomogenizer at a temperature of 20° C.

Subsequently optionally other ingredients, not being lipid, may beadded.

4 Sterilization

Subsequently, the emulsion obtained in step 3 is preferably sterilized.Preferably sterilization takes place in-line at ultra high temperature(UHT) and/or in appropriate containers to obtain a formula in the formof a sterile liquid. A suitable way for UHT treatment is a treatment at120-130° C. for at least 20 s. Alternatively this sterilization step 4is performed before the homogenization step 3.

Preferably the composition obtained by the above process is spray driedafterwards.

Alternatively, the emulsion obtained in step 3 is concentrated byevaporation, subsequently sterilized at ultra high temperature andsubsequently spray dried to give a spray dried powder which is filledinto appropriate containers.

The difference on coating of the lipid globules can further be derivedfrom the zeta potential. Zeta potential (ζ potential) measures thedifference in milliVolts (mV) in electrokinetic potential between thetightly bound layer around the surface and the distant zone ofelectroneutrality and is a measure of the magnitude of the repulsion orattraction between particles in a dispersion. Its value is also relatedto the stability of colloidal dispersions. A high absolute zetapotential will confer stability, i.e. the solution or dispersion willresist aggregation.

Lipid Globule Size

According to the present invention, lipid is present in the compositionin the form of lipid globules, emulsified in the aqueous phase. Thelipid globules comprise a core and a coating. The core comprisesvegetable fat and preferably comprises at least 90 wt. % triglyceridesand more preferably essentially consists of triglycerides. The coatingcomprises phospholipids and/or polar lipids. Not all phospholipidsand/or polar lipids that are present in the composition need necessarilybe comprised in the coating, but preferably a major part is. Preferablymore than 50 wt. %, more preferably more than 70 wt, %, even morepreferably more than 85 wt. %, most preferably more than 95 wt. % of thephospholipids and/or polar lipids that are present in the compositionare comprised in the coating of lipid globules. Not all vegetable lipidsthat are present in the composition need necessarily be comprised in thecore of lipid globules, but preferably a major part is, preferably morethan 50% wt. %, more preferably more than 70 wt. %, even more preferablymore than 85 wt. %, even more preferably more than 95 wt. %, mostpreferably more than 98 wt. % of the vegetable lipids that are presentin the composition are comprised in the core of lipid globules.

In one embodiment the lipid globules of the present invention preferablyhave

1. a volume-weighted mode diameter above 1.0 μm, preferably above 3.0μm, more preferably 4.0 μm or above, preferably between 1.0 and 10 μm,more preferably between 2.0 and 8.0 μm, even more preferably between 3.0and 8.0 μm, most preferably between 4.0 μm and 8.0 μm and/or2. a size distribution in such a way that at least 45 volume %,preferably at least 55 volume %, even more preferably at least 65 volume%, even more preferably at least 75 volume % has a diameter between 2and 12 μm. More preferably at least 45 volume %, preferably at least 55volume %, even more preferably at least 65 volume %, even morepreferably at least 75 volume % has a diameter between 2 and 10 μm. Evenmore preferably at least 45 volume %, preferably at least 55 volume %,even more preferably at least 65 volume %, even more preferably at least75 volume % has a diameter between 4 and 10 μm.

In another preferred embodiment the lipid globules of the presentinvention preferably have

1. a volume-weighted mode diameter below 1.0 μm, and preferably in therange of 0.2-0.7 μm, more preferably in the range of 0.3-0.6 μm, and

2. a size distribution in such a way that less than 45 volume %, has adiameter between 2 and 12 μm, preferably a size distribution whereinmore than 55 volume % of the lipid globules has a diameter of less than2 μm, more preferably a size distribution in such a way that less than35 volume %, has a diameter between 2 and 12 μm, even more preferably asize distribution wherein more than 65 volume % of the lipid globuleshas a diameter of less than 2 μm.

The percentage of lipid globules is based on volume of total lipid. Themode diameter relates to the diameter which is the most present based onvolume of total lipid, or the peak value in a graphic representation,having on the X—as the diameter and on the Y—as the volume (%).

The volume of the lipid globule and its size distribution can suitablybe determined using a particle size analyzer such as a Mastersizer(Malvern Instruments, Malvern, UK), for example by the method describedin Michalski et al, 2001, Lait 81: 787-796.

Digestible Carbohydrate Component

The composition preferably comprises digestible carbohydrate. Thedigestible carbohydrate preferably provides 30 to 80% of the totalcalories of the composition. Preferably the digestible carbohydrateprovides 40 to 60% of the total calories. When in liquid form, e.g. as aready-to-feed liquid, the composition preferably comprises 3.0 to 30 gdigestible carbohydrate per 100 ml, more preferably 6.0 to 20, even morepreferably 7.0 to 10.0 g per 100 ml. Based on dry weight the presentcomposition preferably comprises 20 to 80 wt. %, more preferably 40 to65 wt. % digestible carbohydrates.

Preferred digestible carbohydrate sources are lactose, glucose, sucrose,fructose, galactose, maltose, starch and maltodextrin. Lactose is themain digestible carbohydrate present in human milk. The presentcomposition preferably comprises lactose. The present compositionpreferably comprises digestible carbohydrate, wherein at least 35 wt. %,more preferably at least 50 wt. %, more preferably at least 75 wt. %,even more preferably at least 90 wt. %, most preferably at least 95 wt.% of the digestible carbohydrate is lactose. Based on dry weight thepresent composition preferably comprises at least 25 wt. % lactose,preferably at least 40 wt. %.

Non-Diqestible Oliqosaccharides

Preferably the present composition comprises non-digestibleoligosaccharides with a degree of polymerization (DP) between 2 and 250,more preferably 3 and 60. The non-digestible oligosaccharidesadvantageously improve intestinal microbiota.

The non-digestible oligosaccharide is preferably selected from the groupconsisting of fructo-oligosaccharides (such as inulin),galacto-oligosaccharides (such as transgalacto-oligosaccharides orbeta-galacto-oligisaccharides), gluco-oligosaccharides (such as gentio-,nigero- and cyclodextrin-oligosaccharides), arabino-oligosaccharides,mannan-oligosaccharides, xylo-oligosaccharides, fuco-oligosaccharides,arabinogalacto-oligosaccharides, glucomanno-oligosaccharides,galactomanno-oligosaccharides, sialic acid comprising oligosaccharidesand uronic acid oligosaccharides. Preferably the composition comprisesgum acacia on combination with a non-digestible oligosaccharide.

Preferably the present composition comprises fructo-oligosaccharides,galacto-oligosaccharides and/or galacturonic acid oligosaccharides, morepreferably galacto-oligosaccharides, most preferablytransgalacto-oligosaccharides. In a preferred embodiment the compositioncomprises a mixture of transgalacto-oligosaccharides andfructo-oligosaccharides. Preferably the present composition comprisesgalacto-oligosaccharides with a DP of 2-10 and/orfructo-oligosaccharides with a DP of 2-60. The galacto-oligosaccharideis preferably selected from the group consisting oftransgalacto-oligosaccharides, lacto-N-tetraose (LNT),lacto-N-neotetraose (neo-LNT), fucosyl-lactose, fucosylated LNT andfucosylated neo-LNT. In a particularly preferred embodiment the presentmethod comprises the administration of transgalacto-oligosaccharides([galactose]_(n)-glucose; wherein n is an integer between 1 and 60, i.e.2, 3, 4, 5, 6, . . . , 59, 60; preferably n is selected from 2, 3, 4, 5,6, 7, 8, 9, or 10). Transgalacto-oligosaccharides (TOS) are for examplesold under the trademark Vivinal™ (Borculo Domo Ingredients,Netherlands). Preferably the saccharides of thetransgalacto-oligosaccharides are β-linked.

Fructo-oligosaccharide is a non-digestible oligosaccharide comprising achain of β linked fructose units with a DP or average DP of 2 to 250,more preferably 10 to 100. Fructo-oligosaccharide includes inulin, levanand/or a mixed type of polyfructan. An especially preferredfructo-oligosaccharide is inulin. Fructo-oligosaccharide suitable foruse in the compositions is also already commercially available, e.g.Raftiline®HP (Orafti).

Uronic acid oligosaccharides are preferably obtained from pectindegradation. Uronic acid oligosaccharides are preferably galacturonicacid oligosaccharides. Hence the present composition preferablycomprises a pectin degradation product with a DP between 2 and 100.Preferably the pectin degradation product is prepared from apple pectin,beet pectin and/or citrus pectin. Preferably the composition comprisestransgalacto-oligosaccharide, fructo-oligosaccharide and a pectindegradation product. The weight ratiotransgalacto-oligosaccharide:fructo-oligosaccharide:pectin degradationproduct is preferably (20 to 2):1:(1 to 3), more preferably (12 to7):1:(1 to 2).

Preferably, the composition comprises of 80 mg to 2 g non-digestibleoligosaccharides per 100 ml, more preferably 150 mg to 1.50 g, even morepreferably 300 mg to 1 g per 100 ml. Based on dry weight, thecomposition preferably comprises 0.25 wt. % to 20 wt. %, more preferably0.5 wt. % to 10 wt. %, even more preferably 1.5 wt. % to 7.5 wt. %. Alower amount of non-digestible oligosaccharides will be less effectivein providing a beneficial prebiotic effect, whereas a too high amountwill result in side-effects of bloating and abdominal discomfort.

Protein Component

The present composition preferably comprises proteins. The proteincomponent preferably provides 5 to 15% of the total calories. Preferablythe present composition comprises a protein component that provides 6 to12% of the total calories. More preferably protein is present in thecomposition below 9% based on calories, more preferably the compositioncomprises between 7.2 and 8.0% protein based on total calories, evenmore preferably between 7.3 and 7.7% based on total calories. Theprotein concentration in a nutritional composition is determined by thesum of protein, peptides and free amino acids. Based on dry weight thecomposition preferably comprises less than 12 wt. % protein, morepreferably between 9.6 to 12 wt. %, even more preferably 10 to 11 wt. %.Based on a ready-to-drink liquid product the composition preferablycomprises less than 1.5 g protein per 100 ml, more preferably between1.2 and 1.5 g, even more preferably between 1.25 and 1.35 g.

The source of the protein should be selected in such a way that theminimum requirements for essential amino acid content are met andsatisfactory growth is ensured. Hence protein sources based on cows'milk proteins such as whey, casein and mixtures thereof and proteinsbased on soy, potato or pea are preferred. In case whey proteins areused, the protein source is preferably based on acid whey or sweet whey,whey protein isolate or mixtures thereof and may include α-lactalbuminand β-lactoglobulin. More preferably, the protein source is based onacid whey or sweet whey from which caseino-glyco-macropeptide (CGMP) hasbeen removed. Removal of CGMP from sweet whey protein advantageouslyreduces the threonine content of the sweet whey protein. A reducedthreonine content is also advantageously achieved by using acid whey.Optionally the protein source may be supplemented with free amino acids,such as methionine, histidine, tyrosine, arginine and/or tryptophan inorder to improve the amino acid profile. Preferably α-lactalbuminenriched whey protein is used in order to optimize the amino acidprofile. Using protein sources with an optimized amino acid profilecloser to that of human breast milk enables all essential amino acids tobe provided at reduced protein concentration, below 9% based oncalories, preferably between 7.2 and 8.0% based on calories and stillensure a satisfactory growth. If sweet whey from which CGMP has beenremoved is used as the protein source, it is preferably supplemented byfree arginine in an amount of from 0.1 to 3 wt. % and/or free histidinein an amount of from 0.1 to 1.5 wt. % based on total protein.

Preferably the composition comprises at least 3 wt. % casein based ondry weight. Preferably the casein is intact and/or non-hydrolyzed.

Nutritional Composition

The present composition is preferably particularly suitable forproviding the daily nutritional requirements to a human with an agebelow 36 months, particularly an infant with the age below 24 months,even more preferably an infant with the age below 18 months, mostpreferably below 12 months of age. Hence, the nutritional composition isfor feeding or is used for feeding a human subject. The presentcomposition comprises a lipid, and preferably a protein and preferably adigestible carbohydrate component wherein the lipid component preferablyprovides 30 to 60% of total calories, the protein component preferablyprovides 5 to 20%, more preferably 5 to 15 wt. %, of the total caloriesand the digestible carbohydrate component preferably provides 25 to 75%of the total calories. Preferably the present composition comprises alipid component providing 35 to 50% of the total calories, a proteincomponent provides 6 to 12% of the total calories and a digestiblecarbohydrate component provides 40 to 60% of the total calories. Theamount of total calories is determined by the sum of calories derivedfrom protein, lipids and digestible carbohydrates.

The present composition is not human breast milk. The presentcomposition comprises vegetable lipids. The compositions of theinvention preferably comprise other fractions, such as vitamins,minerals according to international directives for infant formulae.

In one embodiment the composition is a powder suitable for making aliquid composition after reconstitution with an aqueous solution,preferably with water. Preferably the composition is a powder to bereconstituted with water. It was surprisingly found that the size andthe coating with polar lipids of the lipid globules remained the sameafter the drying step and subsequent reconstitution.

In order to meet the caloric requirements of the infant, the compositionpreferably comprises 50 to 200 kcal/100 ml liquid, more preferably 60 to90 kcal/100 ml liquid, even more preferably 60 to 75 kcal/100 ml liquid.This caloric density ensures an optimal ratio between water and calorieconsumption. The osmolarity of the present composition is preferablybetween 150 and 420 mOsmol/l, more preferably 260 to 320 mOsmol/l. Thelow osmolarity aims to reduce the gastrointestinal stress. Stress caninduce adipocyte formation.

Preferably the composition is in a liquid form, with a viscosity below35 mPa·s, more preferably below 6 mPa·s as measured in a Brookfieldviscometer at 20° C. at a shear rate of 100 s⁻¹. Suitably, thecomposition is in a powdered from, which can be reconstituted with waterto form a liquid, or in a liquid concentrate form, which should bediluted with water. When the composition is in a liquid form, thepreferred volume administered on a daily basis is in the range of about80 to 2500 ml, more preferably about 450 to 1000 ml per day.

Infant

The composition of the present invention is preferably for use ininfants. Because of the benefits for the developing child, it isadvantageous to establish the present fatty acid programming effect ofn3 and n6 (LC-)PUFA incorporation in brain membranes early in life.Hence the present composition is preferably administered to the humansubject during the first 3 years of life. In one embodiment of the useaccording to the present invention, the nutritional composition is forfeeding or is used for feeding a human subject with an age between 0 and36 months. The present composition is advantageously administered to ahuman of 0-24 months, more preferably to a human of 0-18 months, mostpreferably to a human of 0-12 months.

Preferably the composition is to be used in infants which areprematurely born or which are small for gestational age. These infantsexperience after birth a catch up growth, which requires extra attentionon proper fat handling. Preferably the composition is to be used ininfants which are large for gestational age, since in these infantsproper allocation of ingested fat is required.

Application

The present composition is preferably administered orally to the infant.The present invention is preferably considered to be of benefit forcognitive and/or behavioural performances at the age above 36 months andfor the general condition of the brain later in life. In one embodimentthe present method is for achieving the effects described herein whensaid human subject has an age above 36 months, preferably when saidhuman subject has an age above 5 years, particularly above 13 years,more particularly above 18 years. In one embodiment the present methodor the present nutritional composition is for feeding a human subjectwith an age between 0 and 36 months and for achieving the effectsdescribed herein when said human subject has an age above 36 months,preferably when said human subject has an age above 5 years,particularly above 13 years, more particularly above 18 years. In oneembodiment the present method is for altering brain membrane fatty acidcomposition selected from the group consisting of increasing brainmembrane fluidity, increasing brain membrane PUFA, increasing brainmembrane LC-PUFA, increasing brain membrane n3 PUFA, increasing brainmembrane n3 LC-PUFA, increasing brain membrane DHA of a human subject,decreasing the ratio of brain membrane n6/n3 (LC-)PUFA, amelioration ofcognitive performance preferably memory performance and/or languagedevelopment performance, more preferably learning memory performance,behavioural performance, visual acuity and fine motor skills, in a humansubject, when said human subject has an age above 36 months, preferablywhen said human subject has an age above 5 years, particularly above 13years, more particularly above 18 years. In one embodiment the presentmethod or the present nutritional composition is for feeding a humansubject with an age between 0 and 36 months and for altering brainmembrane fatty acid composition selected from the group consisting ofincreasing brain membrane fluidity, increasing brain membrane PUFA,increasing brain membrane LC-PUFA, increasing brain membrane n3 PUFA,increasing brain membrane n3 LC-PUFA, increasing brain membrane DHA,decreasing ratio of n6/n3 brain membrane (LC-)PUFA of a human subject,amelioration of cognitive performance, preferably memory performanceand/or language development performance, more preferably learning memoryperformance, behavioural performance, visual acuity and fine motorskills, in a human subject, when said human subject has an age above 36months, preferably at the age above 5 years, particularly above 13years, more particularly above 18 years. In one embodiment increasingbrain membrane fluidity, increasing brain membrane PUFA, increasingbrain membrane LC-PUFA, increasing brain membrane n3 PUFA, increasingbrain membrane n3 LC-PUFA, increasing brain membrane DHA, decreasingration of barin membrane n6/n3 (LC-)PUFA and amelioration of cognitiveperformance preferably memory performance and/or language developmentperformance, more preferably learning memory performance, behaviouralperformance, visual acuity, fine motor skills occurs later in life. Withlater in life is meant an age exceeding the age at which the diet istaken, preferably with at least one year.

Cognitive performance in the present invention refers preferably to anyone selected from the group consisting of memory (such as short termmemory, long term memory) performance, learning capacity, alertness,attention, and concentration capacity, more preferably memoryperformance and/or language development performance, more preferablylearning memory performance.

The inventors surprisingly found that when mice during infancy andchildhood were fed a food composition comprising lipid globules coatedwith polar lipids, a different and significant effect on brain membranecomposition later in life was observed compared to mice which duringinfancy and childhood had been fed a food composition having a similarfatty acid composition, but no polar lipids, in particular present inthe form of a coating. At day 42, which is a time point corresponding tochildhood in a human setting, no significant differences were observedin growth (weight) between the groups, but from day 42 both groups werefed a Western style diet which was high in fat. Surprisingly at day 98,which is a time point corresponding to early adulthood in humans, themice, which had previously consumed the food composition of the presentinvention before turning to the Western style diet, had a significantlyincreased amount of brain membrane PUFA, LC-PUFA, and DHA than micewhich had received a control composition. Consequently, the presentfinding can be put to use for prevention and/or treatment of a disorderassociated with decreased brain membrane fluidity and/or associated withdecreased brain membrane PUFA content and/or LC-PUFA content orincreased ratio n6/n3 PUFA. More in particular, the present finding canbe put to use for prevention and/or treatment of a psychiatric,psychological and/or neurobiological disorder. Specific effects thepresent finding which can be expected more early in life reside in theamelioration of visual acuity and/or fine motor skills. Also the presentfinding is of benefit for amelioration of cognitive performance and/orbehavioural performance. To even further specify the benefits of thepresent finding of an for altered fat handling, ultimately resulting inimproved fatty acid availability especially in brain cell membranes, thepresent invention is for the treatment and/or prevention of attentiondeficiency, attention deficit hyperactivity disorder (ADHD), dyslexia,autism, depression, bipolar depression, anxiety, schizophrenia,obsessive compulsive disorder (OCD), bulimia, abuse of alcohol or drugs,borderline personality disorder, panic disorder, social phobia, learningdifficulties, mild cognitive impairment learning memory impairment,language development impairment, dementia, Alzheimer's disease andParkinson's disease. As a non-optimal brain fatty acid composition isconsidered to be very important for developing ADHD, dyslexia andautism, the present invention is preferably for the treatment and/orprevention of one selected from the group consisting of ADHD, dyslexiaand, autism. As a non-optimal brain fatty acid composition is consideredto be very important for developing mild cognitive impairment, learningmemory impairment, dementia, Alzheimer's disease and Parkinson's diseasethe present invention is preferably for the treatment and/or preventionof one selected from the group consisting of mild cognitive impairmentlearning memory impairment, Alzheimer's disease and Parkinson's diseasemore preferably learning memory impairment. The inventors proved thisconcept by demonstrating an improved performance in the Morris watermazetasks by rats having consumed the nutritional composition of the presentinvention.

Though the nutritional composition is in particular suitable forinfants, because of the long term effects and the enhanced plasticity ofthe brain during infancy, the nutritional composition is also suitablefor other human subjects. It is believed that also later in life thebrain membrane fatty acid composition can be adapted. Direct dieteffects on brain membrane fatty acid composition were already observedafter 5 days of administration of the diet of the present invention.Therefore in a preferred embodiment the present nutritional compositioncan also be used in other human subjects, preferably patients sufferingfrom one of the disorders mentioned above and/or elderly. Elderly in thepresent invention are defined as humans with an age of 50 or above, morepreferably 60 or above, even more preferably 65 or above.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition, reference to an element by the indefinitearticle “a” or “an” does not exclude the possibility that more than oneof the element is present, unless the context clearly requires thatthere be one and only one of the elements. The indefinite article “a” or“an” thus usually means “at least one”.

Example 1: Process for Preparing an IMF with Lipid Globules Differing inArchitecture

An infant formula was prepared comprising per kg powder about 4800 kcal,248 g lipid, 540 g digestible carbohydrates, 55 g non-digestibleoligosaccharides and 103 g protein.

The composition was prepared using, a vegetable oil blend, demineralisedwhey powder, lactose, non-digestible oligosaccharides(galacto-oligosaccharides and long chain fructo-oligosaccharides in aweight ratio of 9/1). Vitamins, minerals, and trace elements as known inthe art were used. For diet 3 to 6 also a butter milk serum powdercomprising polar lipids from milk origin was used. An aqueous phase wasprepared mixing all components, besides inulin and the oil blend and fordiet 3 and 5 also the butter milk serum, in water, at room temperature,by stirring. Potassium hydroxide was used to set the pH at 6.8-7.0. Thedry weight matter of the mixture was about 27%. The mixture was heatedto 60° C. The vegetable oil blend was also heated to 60° C. and added tothe water phase and blended with an Ultra-Turrax T50 for about 30-60 sat 5000-10000 rpm.

Subsequently the oil-water mixture was homogenised at a pressure of 100bar in a first step and 50 bar in a second step in a Niro Suavi NS 2006H Homogenizer for diet 1, 3 and 5. For diet 2 and 3 and 4 this mixturewas homogenized in two steps at a pressure of 5 and 20 bar,respectively. The temperature was 60° C. The product was UHT treated at125° C. for 30 s. The product was dried to a powder by spray drying.Long chain inulin was blended dry into the powder. For diet 3 and 4 alsobutter milk serum powder was dry blended into the powder.

The amount of vegetable glycerophospholipids was 0.2 wt. % based ontotal fat for diet 1 and 2. Diet 1 and 2 did not contain sphingolipidsand cholesterol. Diet 3 and 4 comprised about 1.83 wt. %glycerophospholipids based on total fat, of which about 90% derived fromthe butter milk powder and about 10% already present in the standard IMFderived from vegetable oils, and further comprised milk derivedsphingolipids of which the majority (about 0.47 wt. % based on totalfat) was sphingomyelin; the rest being glycosphingolipids. Diet 3 and 4comprised about 0.05 wt. % milk derived cholesterol based on total fat.Diet 5 and 6 comprised half of the amount of milk derived polar lipidsbased on total fat of diet 3 and 4.

The size of the lipid globules was measured with a Mastersizer 20000(Malvern Instruments, Malvern UK) and shown in Table 1. Coating of thelipid globules with polar lipids in diet 5 and 6 and absence of coatingin diet 1, 2, 3 and 4 was confirmed by confocal laser scanningmicroscopy method. It was checked with confocal laser scanningmicroscopy that the larger lipid globules of the present invention werecoated with phospholipids, before spray drying and after reconstitutionof the spray dried powder with water. In both cases the lipid globulesof diet 4 and 6 were covered with a layer of phospholipids. Asfluorescent probes Annexin V Alexa Fluor 488 (In Vitrogen molecularprobes) for labeling the phospholipids, and Nile Red (Sigma-Aldrich) forlabeling triglycerides, were used. After labeling the milk samplesVectrashield mounting medium (Vector laboratories inc., Burliname USA)for reducing particle movement and photo-bleaching was added.Observations were made using a Zeiss Laser Scanning Microscope withexcitation wavelengths of 488/543/633 nm and emission filters set atband pass 505-530, and band pass 560-615. Also the size of the lipidglobules was the same before drying and after reconstitution of thespray dried powder with water.

TABLE 1 Lipid globule characteristics of different milks Volume % Volumewith a diameter Mode between IMF diameter μm 2 and 12 μm 1, Standard IMF(small lipid globules) 0.5 5.1 2, Experimental IMF (large lipidglobules) 4.0 72.2 3, Experimental IMF (small lipid globules, 0.4 3.9free polar lipids) 4, Experimental IMF (large lipid globules, 5.0 74.8free polar lipids) 5, Experimental IMF (small lipid globules, 0.5 4.3coated with polar lipids) 6, Experimental IMF (large lipid globules, 4.370.3 coated with polar lipids)

After 5 months storage at room temperature the size of the lipidglobules in diet 1, 3 and 5 had not changed, with a volume mode diameterof 0.5, 0.4 and 0.5 respectively. Also the volume mode diameter of diet2, 4 and 6 were rather stable being 4.8 μm, 7.9 μm and 6.6 μm,respectively.

Example 2

Offspring of C57/BL6 dams were weaned from day 15 on. The experimentalweaning diets were continued until day 42. From day 42 to day 98 allpups were ad libitum fed the same diet based on AIN-93G diet with anadjusted lipid fraction (containing 10 wt. % lipid of which 50 wt. %lard and 1 cholesterol, based on total lipid), which is representativefor a Western style diet.

The experimental diets that were used for weaning were:

1) an infant milk formula (IMF) based control diet. This diet comprised282 g standard IMF (IMF 1 of example 1) per kg, with the lipid globulesize as mentioned in example 1. The rest of the diet was AIN-93Gprotein, carbohydrates and fibre. All lipid present in the diet wasderived from the IMF.2) an IMF based experimental diet. This diet comprised 282 gexperimental IMF (IMF 2 of example 1) per kg, with the lipid globulesize as mentioned in example 1. The rest of the diet was AIN-93Gprotein, carbohydrates and fibre. All lipid present in the diet wasderived from the IMF.3) an IMF based experimental diet. This diet comprised 282 gexperimental IMF (IMF 3 of example 1) per kg, with the lipid globulesize as mentioned in example 1 and comprising phospholipids in freeform. The rest of the diet was AIN-93G protein, carbohydrates and fibre.All lipid present in the diet was derived from the IMF.4) an IMF based experimental diet. This diet comprised 282 gexperimental IMF (IMF 4 of example 1) per kg, with the lipid globulesize as mentioned in example 1 and comprising phospholipids in freeform. The rest of the diet was AIN-93G protein, carbohydrates and fibre.All lipid present in the diet was derived from the IMF.5) an IMF based experimental diet. This diet comprised 282 gexperimental IMF (IMF 5 of example 1) per kg, with the lipid globulesize as mentioned in example 1 and with phospholipids present as acoating around the lipid globules. The rest of the diet was AIN-93Gprotein, carbohydrates and fibre. All lipid present in the diet wasderived from the IMF.6) an IMF based experimental diet. This diet comprised 282 gexperimental IMF (IMF 6 of example 1) per kg, with the lipid globulesize as mentioned in example 1 and with phospholipids present as coatingaround the lipid globules. The rest of the diet was AIN-93G protein,carbohydrates and fibre. All lipid present in the diet was derived fromthe IMF.

At day 42, all mice switched to a “Western style diet” comprising 4016kJ per 100 g, 10 wt. % lipid, 1 wt % cholesterol based on total fat, 60wt % digestible carbohydrates, 4.75 wt. % fibers, and 17.9 wt. %protein, until day 98.

The fatty acid composition of the experimental diets was very similar inrespect of saturated, mono-unsaturated, poly unsaturated and long chainpoly unsaturated fatty acids, with calculated linoleic acid (LA) of 14wt % in diet 1 and 2, and 13.2 wt % in diet 3, 4, 5 and 6, based ontotal fatty acids, with alpha-linoleic acid (ALA) of 2.6 wt. % in diet 1and 2 and 2.5 wt % in diet 3, 4, 5 and 6, based on total fatty acids andwith LA/ALA ratio of 5.4 in diet 1 and 2 and 5.3 in diet 3, 4, 5 and 6,respectively. The amount of DHA was 0.2 wt. % in all 6 diets, and theamount of ARA was 0.35 wt. % in diet 1 and 2 and 0.36 wt. % in diet 3,4, 5 and 6. In the Western style diet the amount of LA was 11.9 wt. %,the amount of ALA was 1.3 wt. %, based on total fatty acids and theratio LA/ALA was 9.15.

The mice were weighed twice a week. The food intake was determined oncea week during the entire experiment. To determine body composition(i.e., fat mass (FM) and fat-free mass (FFM)) DEXA scans (Dual EnergyX-ray Absorbiometry) were performed under general anesthesia at 6, 10and 14 weeks of age, 42, 70, and 98 days after birth respectively, bydensitometry using a PIXImus imager (GE Lunar, Madison, Wis., USA). Atthe age of 98 days the male mice were sacrificed and organs includingbrains were dissected and weighed. Of each brain, 1 hemisphere washomogenized (Utra-Turrax T25 basic, IKA, VWR international) in 50volumes of ice cold deionized water (MiliQ). Subsequently, brain fattyacid (FA) profile was quantified by means of gas chromatographicanalysis. 1 ml brain homogenate was extracted according to the procedureof Bligh & Dyer (dichloromethane/methanol extraction). The lipids wereconverted into methyl esters with concentrated sulfuric acid inmethanol. The fatty acid methyl esters (FAME) were extracted from themethanol solution with hexane and analyzed on a gas chromatograph (GC)equipped with a flame ionization detector (FID).

Results:

At day 98, the FA profile of the brains was determined. Table 2 showsthe general FA profile of the brains (SFA, MUFA, PUFA, LCPUFA, n3, n6,n6/n3-ratio, n3 LC-PUFA, n6 LC-PUFA, n6/n3 LC-PUFA ratio) and Table 3shows the profile of specific LC-PUFA's (DHA, EPA, ARA, ALA, C22:4 n6,C22:5 n−3 and C22:5 n−6).

When differences between individual programming diets were compared, noeffects of programming diet were found on % SFA and % MUFA, but the % ofall other parameters in the FA profile were affected; PUFA, LC-PUFA, n6PUFA's, n3 PUFA's, n6/n3 PUFA ratio, n6 LC-PUFA's, n3 LC-PUFA's, andn6/n3 LC-PUFA ratio. Many of these effects were related to the somewhatdifferent brain FA profile of animals that were raised on the largelipid globule diet without phospholipids (diet 2) compared to the otherdiets. Diet 2 resulted in lower PUFA, LC-PUFA, n6 PUFA, n3 PUFA, n6 andn3 LC-PUFA than the other diets (p<0.05).

TABLE 2 Fatty acid composition of brain membranes later in life after anearly diet with different lipid globules Diet Diet 5 Diet 6 Diet 3 Diet4 Small lipid Large lipid Diet 1 Diet 2 Small lipid Large lipidglobules, globules, Small lipid Large lipid globules, free globules,free coated with coated with globules globules polar lipids polar lipidspolar lipids polar lipids SFA 40.23 ± 0.39 41.17 ± 0.46 40.43 ± 0.5040.06 ± 0.48 40.25 ± 0.42 40.06 ± 0.35 MUFA 24.25 ± 0.37 25.09 ± 0.4424.11 ± 0.40 24.91 ± 0.37 24.31 ± 0.61 23.81 ± 0.54 PUFA 25.79 ± 0.1724.59 ± 0.38 26.02 ± 0.33 25.59 ± 0.23 25.75 ± 0.56 26.34 ± 0.35 LC-PUFA24.53 ± 0.19 23.40 ± 0.39 24.92 ± 0.34 24.42 ± 0.24 24.61 ± 0.58 25.16 ±0.38 n6 11.29 ± 0.13 10.89 ± 0.12 11.53 ± 0.16 11.19 ± 0.12 11.34 ± 0.1911.48 ± 0.20 n3 14.40 ± 0.10 13.59 ± 0.28 14.24 ± 0.22 14.18 ± 0.1414.19 ± 0.28 14.64 ± 0.17 n6/n3  0.78 ± 0.010  0.80 ± 0.011  0.81 ±0.013  0.79 ± 0.008  0.80 ± 0.009  0.78 ± 0.010 n6 LC 10.60 ± 0.11 10.30± 0.12 10.99 ± 0.15 10.61 ± 0.12 10.77 ± 0.27 10.84 ± 0.21 n3 LC 13.83 ±0.12 12.99 ± 0.29 13.70 ± 0.24 13.59 ± 0.15 13.63 ± 0.30 14.10 ± 0.20n6/n3 LC  0.77 ± 0.007  0.80 ± 0.012  0.80 ± 0.014  0.78 ± 0.007  0.79 ±0.007  0.77 ± 0.010

Furthermore, the n6/n3 ratio in the brains of diet 3 fed animals washigher than diet 1 (p=0.043) (PL effect) and diet 4 (p=0.083) (sizeeffect). The n6/n3 LC-PUFA ratio was higher in the brains of the animalsthat received the diet 5 diet compared to diet 1 (p=0.082) (PLcoatingeffect) and the diet 6 (p=0.093) (size effect).

The % of the specific LCPUFA's DHA, EPA, ARA, ALA, C22:4 n−6, C22:5 n−3and C22:5 n−6 is depicted in Table 3. LA was not detected in the brain.

There was no effect of programming diet on % of ALA, ARA and C22:5 n3.The % of C22:5 n6 was affected by programming diet (p<0.001), the % ofC22:5 n6 was lower in the diet 1 and diet 2 groups than in the groupswith diet 3-6 (p<0.001), which emphasizes that adding PL to the dietresults in higher % C22:5 n6. There was also an effect of programmingdiet on % of C22:4 n6 (p=0.003), the % of C22:4 n6 was higher in animalsfrom the diet 3 group than from the diet 1 group (p=0.059 trend) anddiet 4 group (p=0.061 TREND). The % of DHA was affected by diet as well(p=0.038), the % of DHA in the brains of animals in the diet 2 group waslower than that of diet 1 (p=0.008), diet 4 (p=0.086, trend) and diet 6(p=0.001), the diet 6 group was also higher than diet 4 (p=0.091,trend). For the % of EPA, a significant effect of diet was also present(p=0.050), the % of EPA was lower in the diet 1 group than in the diet 3group (z=−1.815, p=0.069 TREND), diet 4 group (p=0.033), diet 5 group(p=0.029) and diet 6 group (p=0.074 trend) group. There was also adifference in the % of EPA between the diet 2 group and diet 4 group(p=0.050), these effects emphasize the previously described effect of PLin the diet on EPA.

TABLE 3 Diet Diet 5 Diet 6 Diet 3 Diet 4 Small lipid Large lipid Diet 1Diet 2 Small lipid Large lipid globules, globules, Small lipid Largelipid globules, free globules, free coated with coated with globulesglobules polar lipids polar lipids polar lipids polar lipids C18:3 n3(ALA) 0.47 ± 0.02 0.49 ± 0.02 0.45 ± 0.02 0.48 ± 0.02 0.46 ± 0.03 0.44 ±0.03 C18:2 n6 LA C20:4 n6 (ARA) 7.62 ± 0.12 7.40 ± 0.12 7.86 ± 0.13 7.58± 0.11 7.66 ± 0.22 7.71 ± 0.18 C20:5 n3 (EPA) 0.006 ± 0.003 0.006 ±0.003 0.021 ± 0.008 0.032 ± 0.011 0.023 ± 0.007 0.016 ± 0.005 C22:6 n3(DHA) 13.40 ± 0.13  12.52 ± 0.30  13.25 ± 0.24  13.08 ± 0.15  13.17 ±0.32  13.64 ± 0.12  C22:4 n6 (DTA) 2.22 ± 0.03 2.20 ± 0.02 2.30 ± 0.032.23 ± 0.02 2.29 ± 0.04 2.23 ± 0.04 C22:5 n6 (“DPA”) 0.04 ± 0.01 0.05 ±0.01 0.19 ± 0.01 0.17 ± 0.02 0.17 ± 0.01 0.17 ± 0.01 C22:5 n3 (DPA) 0.14 ± 0.019  0.16 ± 0.006  0.15 ± 0.008  0.16 ± 0.004  0.15 ± 0.005 0.17 ± 0.005

In a strategy to use large lipid globules for achieving an effect onobesity later in life, it was found that brain membrane fatty acidcomposition was not improved compared to standard infant milk formula.Also standard IMF in terms of lipid globule size wherein the lipidglobules had a phospholipid coating showed no improvement in brainmembrane fatty acid composition. However, only in the case of usinglarge lipid globules, coated with phospholipids or polar lipids, animprovement in brain membrane fatty acid composition in terms of(LC-)PUFA content was observed, while the advantageous effect on obesitylater in life was also achieved as well as the advantageous effect onbone mineral accretion.

Thus advantageously the diet with large lipid globules coated withphospholipids and/or polar lipids further showed strong improved effectson long term effects of obesity, visceral adiposity, lean body mass,bone mineral content and bone mineral density compared with a dietcomprising small lipid globules covered mainly with protein.

Overall, the FA profile in the brains of mice exposed early in life todiet 6 with large lipid globules coated with polar lipids, was the bestwith the highest % of PUFA (both n3 and n6) and LC-PUFA (both n3 andn6), and thus an improved fluidity, and a relatively low n6/n3 PUFA andlow n6/n3 LC-PUFA ratio. These effects were especially prominentcompared to large lipids without PL (diet 2). A diet with large lipidglobules with free PL (diet 4) showed intermediate effects, indicatingthat the location of the PL as a coating around the lipid globule playsa role. When small lipid globules were used these effects were much lessclear. No effect of free PL and coating was observed with free PL or PLcoating in small lipid globules and due to slightly increased n6(LC)-PUFA in the presence of PL n6/n3 (LC)-PUFA ratios were slightlyincreased in the presence of PL, which is not desired. Furthermore, ofthe n3-PUFA in diet group 6 beneficially had the highest amount of DHAand DPA, with relatively lower amounts of EPA and ALA.

It can be concluded that both the lipid globules have to be increased insize and they have to be surrounded by a coating comprisingphospholipids in order to see improved long term effect on brain FAcomposition compared to lipid globules as present in standard IMF.

Example 3: Effect of Different Dietary Lipid Globules on Long Term FattyAcid Composition of Brain Membranes

The experiment of example 2 was repeated in a similar way. Difference inlipid globule size was obtained by a difference in homogenizationpressure as described in example 1. The pressure of homogenizatiom was10/5 for diet 2, 4 and 6 and 550/50 for diet 1, 3, and 5. The volumemode diameter of the lipid globules of diet 1, 3, and 5 ranged from 0.23to 0.28 μm. Less than 10 vol. % had a diameter between 2 and 12 μm. Thevolume mode diameter of the lipid globules in diet 2, 4 and 5 rangedfrom 3.0 to 4.4 μm. More than 50 vol. % had a diameter between 2 and 12μm. The source of phospholipids for diet 3 to 6 was SM2 powder fromCorman which was used at a final concentration of about 1.3%phospholipid based on total lipid. Diets were similar as in example 2,except for the Western style diet, which was more unhealthy, comprising4520 kJ per 100 g, 20 wt. % lipid, 1 wt % cholesterol based on totalfat, 52 wt % digestible carbohydrates, 4.75 wt. % fibers, and 17.9 wt. %protein, until day 98.

The fatty acid composition of the experimental diets was very similar inrespect of saturated, mono-unsaturated, poly unsaturated and long chainpoly-unsaturated fatty acids and similar to example 2.

At the age of 98 days the brain membrane fatty acid composition wasdetermined as in example 2. Also on day 98 blood samples were taken andthe fatty acid composition of the red blood cell membranes wasdetermined.

Results:

At day 98, the fatty acid profile of the brains was determined. Table 4shows the general fatty acid profile of the brains and shows the profileof the major LC-PUFA.

TABLE 4 Fatty acid composition of brain membranes later in life after anearly diet with different lipid globules Diet Diet 5 Diet 6 Small lipidLarge lipid globules, globules, Diet 1 coated with coated with Smalllipid globules polar lipids polar lipids PUFA 26.66 (0.15) 26.86 (0.19)26.98 (0.22) LC-PUFA 25.51 (0.16) 25.74 (0.21) 25.88 (0.23) n6 12.15(0.07) 12.29 (0.10) 12.25 (0.13) n3 14.42 (0.12) 14.48 (0.12) 14.64(0.13) n6/n3 0.843 (0.01) 0.849 (0.01) 0.837 (0.01) n6 LC 11.60 (0.06)11.75 (0.10) 11.72 (0.13) n3 LC 13.82 (0.13) 13.90 (0.13) 14.08 (0.14)n6/n3 LC 0.840 (0.01) 0.846 (0.01) 0.833 (0.01) C20:4 n6 (ARA)  8.21(0.07)  8.34 (0.09)  8.33 (0.11) C22:6 n3 (DHA) 13.36 (0.14) 13.45(0.14) 13.64 (0.15) C22:4 n6 (DTA)  2.42 (0.02)  2.42 (0.02)  2.41(0.04)

When differences between individual diets were compared, the amount ofPUFA, LC-PUFA, n6 PUFA's, n3 PUFA's, n6 LC-PUFA's, n3 LC-PUFA's wasincreased in diet 5 and 6 (small and large, phospholipid coated lipidglobules), with diet 6 having the most improved effects on increase ofPUFA, LC-PUFA, n3 PUFA and n3-LC-PUFA. Additionally the ratio of n6/n3PUFA and n6/n3 LC-PUFA was beneficially decreased most in diet 6. Anincrease in DHA and ARA is observed in diet 5 and 6. The amount of DHAin diet 6 is beneficially even further increased compared to diet 5.

No effect in fatty acid composition of the red blood cell membranes wasobserved (data not shown). Since the fatty acid composition is areflection of the fatty acid composition in the diet, this is indicativefor the specific long term effects of early diet on brain membranecomposition. This effect is still present even after a long period on aless healthy diet with low LC-PUFA and a high n6/n3 PUFA ratio.

Example 4: Direct Diet Effects with Different Lipid Globules on FattyAcid Composition of Brain Membranes

Mice and diets similar to example 2 were used. The diets wereadministered from day 15 to day 28. On day 28 the fatty acid compositionof the brain membranes was determined, similar as in example 2.

In this experiment the direct diet effects on brain membrane fatty acidcomposition was determined. The diet was similar as in example 2. As asource of phospholipids 80% butter milk serum (Cormann SN2), and 20% soylecithin was used. The amount of phospholipids added based on total fatwas 2.9 wt. %

The volume mode diameter of the lipid globules of diet 1, 3, and 5ranged from 0.49 to 0.60 μm. Less than 5 vol. % had a diameter between 2and 12 μm. The volume mode diameter of the lipid globules in diet 2, 4and 5 ranged from 4.4 to 7.9 μm. More than 55 vol. % had a diameterbetween 2 and 12 μm.

The results on brain membrane fatty acid composition are shown in areshown in table 5.

TABLE 5 Fatty acid composition of brain membranes after a diet withdifferent lipid globules Diet Diet 5 Diet 6 Diet 3 Small lipid Largelipid Small lipid globules, globules, Diet 1 globules, coated coatedSmall lipid free polar with polar with polar globules lipids lipidslipids PUFA 27.73 (0.20) 27.86 (0.14) 27.89 (0.18) 27.68 (0.16) LC-PUFA26.71 (0.22) 26.91 (0.17) 26.86 (0.19) 26.69 (0.19) n6 13.80 (0.11)13.63 (0.10) 13.56 (0.14) 13.41 (0.11) n3 13.85 (0.15) 14.15 (0.10)14.22 (0.08) 14.19 (0.10) n6/n3  1.00 (0.01) 0.963 (0.01) 0.954 (0.01)0.945 (0.01) n6 LC 13.25 (0.12) 13.12 (0.10) 13.02 (0.15) 12.90 (0.12)n3 LC 13.38 (0.16) 13.70 (0.11) 13.75 (0.09) 13.71 (0.11) n6/n3 LC  0.99(0.01)  0.96 (0.01)  0.95 (0.01)  0.94 (0.01) DHA 12.94 (0.18) 13.31(0.11) 13.33 (0.09) 13.22 (0.12) ARA  9.45 (0.11)  9.40 (0.10)  9.27(0.13)  9.25 (0.13) DTA  2.40 (0.03)  2.37 (0.03)  2.32 (0.03)  2.29(0.02)

As can be deduced from the results, fatty acid composition in brainmembranes was improved after consumption of a diet with lipid globuleswere coated with phospholipids, see effects of diet 5 versus diet 1 onincrease in (LC-)PUFA, DHA and decrease in ARA and n6/3 ratio of (LC)PUFA. This effect was less, when the phospholipids were separatelypresent, not in the form of a coating (see diet 3). This is anindication that the phospholipids should be present in the coating ofthe lipid globule. This effect was further improved using large coatedlipid globules (diet 6 versus diet 5). This is an indication that thecoated lipid globules should preferably be large.

Example 5: Direct Diet Effects of Different Lipid Globules of on FattyAcid Composition of Brain Membranes

Diet 1 and 6 similar to example 2, 3 and 4 were consumed for 5 days byWistar rat pups (day 16-21 after birth). The amount of phospholipids was1.6 wt. % based on total fat. As a source of phospholipids butter milkpowder was used. The mode diameter of the lipid globules in diet 1 was0.5 μm and of diet 6 3.0 μm.

The membrane fatty acid composition of the brain was determined as inexample 2. The results are shown in Table 6.

TABLE 6 Fatty acid composition of brain membranes after 5 days of a dietwith different lipid globules Diet 1 Diet 6 Small uncoated Large coatedlipid globules lipid globules PUFA 30.48 (0.26) 30.53 (0.18) LC-PUFA29.13 (0.26) 29.15 (0.18) n6 PUFA 17.45 (0.22) 17.22 (0.10) n3 PUFA12.98 (0.17) 13.26 (0.09) n6/n3 PUFA  1.35 (0.03)  1.30 (0.01)* n6LC-PUFA 16.45 (0.21) 16.17 (0.10) n3 LC-PUFA 12.64 (0.17) 12.92 (0.09)n6/n3 LC-PUFA  1.30 (0.03)   1.25 (0.01)** DHA 12.27 (0.17)  12.56(0.10)* ARA 11.08 (0.13) 10.83 (0.09) DTA  3.41 (0.06)  3.33 (0.04) *p >0.1, **p > 0.05

As can be deduced from the results, even after 5 days consumption of thenutritional composition of the present invention an improved effect wasobserved on (LC-)PUFA, DHA, and n6/n3 (LC)-PUFA ratio in brainmembranes. The amount of n6(LC-)PUFA advantageously decreased.

Example 6: Effect on Cognitive Performance of a Diet with DifferentLipid Globules

The same diet of example 4 was used. This diet was given to Wistar rats.The male rats consumed the feed from day 16 to day 42. On day 42 themale rats were submitted to a a short version of the Morris water mazetest. Morris water maze tests, also known as Morris water navigationtasks, are widely used in the art of behavioral neuroscience, and is abehavioral procedure to study cognitive function, in particular spatiallearning and memory. In this experiment the animals were trained tolocate a hidden platform based on environmental cues. In short aplatform was submerged just below water surface at a fixed position in around water tank. The tank was in a room with fixed cues on the wallsthat were visible from the water surface in the tank. Animals wereallowed to sit on the submerged platform for 60 seconds to getacquainted with the environment. Directly after that, animals wereplaced in the water tank and had to locate the submerged platform within60 seconds. Animals were trained in 4 successive trials with a differentstart-location each trial. The start location per trial was similar forall animals. Time to locate the platform per trial was measured. Theresults are shown in Table 7.

TABLE 7 Time to reach the platform in Morris water maze task (latency inseconds). diet trial 1 trial 2 trial 3 trial 4 average Diet 1 50.09 ±5.21 40.97 ± 8.78 30.15 ± 8.91 28.88 ± 5.43 37.52 ± 3.50 Diet 6 54.05 ±3.93 27.97 ± 6.40 28.71 ± 8.68 24.98 ± 8.19 33.92 ± 3.49

As can be deduced from table 7, the performance is improved in ratshaving consumed the composition of the present invention. Especiallyprominent was the learning curve, which was much steeper when a diet ofthe present invention is consumed. This is indicative for an improvedcognitive performance, in particular memory performance, more particularlearning memory performance, memory and spatial learning.

The invention claimed is:
 1. A method for amelioration of anxiety orsociability in a human subject in need thereof, the method comprisingadministering to the human subject 80 to 2500 ml daily for at least 5days a nutritional composition comprising: (a) 10 to 50 wt. % vegetablelipids based on dry weight of the composition, (b) 0.5 to 10 wt. %phospholipids based on total lipid, (c) glycosphingolipids and (d)cholesterol, wherein the composition comprises lipid globules with acore comprising the vegetable lipids and a coating comprising thephospholipids, the lipid globules having: (i) a volume-weighted modediameter between 1.0 and 10 pm, and/or (ii) a diameter of 2 to 12 pm inan amount of at least 45 volume % based on total lipid, wherein the corecomprises at least 90 wt. % triglycerides having fatty acids and thecomposition comprises glycerophospholipids having fatty acids derivedfrom milk, and wherein the composition comprises linoleic acid (LA) inan amount between 5 and 15 wt. % based on total fatty acid.
 2. Themethod according to claim 1, wherein the lipid globules have a diameterof 2 to 12 μm in an amount of at least 55 volume % based on total lipid.3. The method according to claim 2, wherein the lipid globules have: (i)a volume-weighted mode diameter between 3.0 and 8.0 μm, and/or (ii) adiameter of 2 to 12 μm in an amount of at least 65 volume % based ontotal lipid.
 4. The method according to claim 1, wherein the compositioncomprises n3 LC-PUFA in an amount of at least 0.2 wt. % based on totalfatty acid and that does not exceed 15 wt. % based on total fatty acid.5. The method according to claim 1, wherein the composition comprisesDHA in an amount of 0.1 to 0.6 wt. % based on total fatty acid.
 6. Themethod according to claim 1, wherein the composition comprises n6LC-PUFA in an amount of at least 0.02 wt. % based on total fatty acidand that does not exceed 5 wt. % based on total fatty acid.
 7. Themethod according to claim 1, wherein the composition comprises ARA in anamount of 0.1 to 0.6 wt. % based on total fatty acid.
 8. The methodaccording to claim 1, wherein the nutritional composition compriseslinoleic acid (LA) and alpha-linolenic acid (ALA) in a weight ratioLA:ALA between 4:1 and 7:1.
 9. The method according to claim 1, whereinthe administration occurs when the human subject is between 0 and 36months of age.
 10. The method according to claim 9, wherein the anxietyor sociability is ameliorated when the human subject has reached an ageabove 36 months.